The importance of PUFAs is undisputed. For example, certain PUFAs are important biological components of healthy cells and are recognized as: “essential” fatty acids that cannot be synthesized de novo in mammals and instead must be obtained either in the diet or derived by further desaturation and elongation of linoleic acid (LA; 18:2 ω-6) or α-linolenic acid (ALA; 18:3 ω-3); constituents of plasma membranes of cells, where they may be found in such forms as phospholipids or triacylglycerols; necessary for proper development (particularly in the developing infant brain) and for tissue formation and repair; and, precursors to several biologically active eicosanoids of importance in mammals (e.g., prostacyclins, eicosanoids, leukotrienes, prostaglandins). Additionally, a high intake of long-chain ω-3 PUFAs has cardiovascular protective effects (Dyerberg, J. et al., Amer. J. Clin. Nutr., 28:958-966 (1975); Dyerberg, J. et al., Lancet, 2(8081):117-119 (Jul. 15, 1978); Shimokawa, H., World Rev. Nutr. Diet, 88:100-108 (2001); von Schacky, C. and Dyerberg, J., World Rev. Nutr. Diet, 88:90-99 (2001)). Numerous other studies document wide-ranging health benefits conferred by administration of ω-3 and/or ω-6 PUFAs against a variety of symptoms and diseases (e.g., asthma, psoriasis, eczema, diabetes, cancer).
Today, a variety of different hosts including plants, algae, fungi and yeast are being investigated as means for commercial PUFA production. Although the natural PUFA-producing abilities of the host organisms are sometimes specific to a given methodology, genetic engineering has also proven that the natural abilities of some hosts (even those natively limited to LA and ALA fatty acid production) can be substantially enhanced to produce high-levels of various long-chain ω-3/ω-6 PUFAs. Whether this effect is the result of natural abilities or recombinant technology, production of arachidonic acid (ARA; 20:4 ω-6), eicosapentaenoic acid (EPA; 20:5 ω-3) and docosahexaenoic acid (DHA; 22:6 ω-3) all require expression of either the Δ9 elongase/Δ8 desaturase pathway (which operates in some organisms, such as euglenoid species and which is characterized by the production of eicosadienoic acid [EDA; 20:2 ω-6] and/or eicosatrienoic acid [ETrA; 20:3 ω-3]) or the Δ6 desaturase/Δ6 elongase pathway (which is predominantly found in algae, mosses, fungi, nematodes and humans and which is characterized by the production of gamma-linoleic acid [GLA; 18:3 ω-6] and/or stearidonic acid [STA; 18:4 ω-3]) (FIG. 1).
For the purposes herein, the present application focuses on use of the Δ9 elongase/Δ8 desaturase pathway, and more specifically, on the use of Δ9 elongase enzymes. Most Δ9 elongase enzymes identified so far have the ability to convert both LA to EDA and ALA to ETrA (wherein DGLA and ETA are subsequently synthesized from EDA and ETrA, respectively, following reaction with a Δ8 desaturase; ARA and EPA are subsequently synthesized from DGLA and ETA, respectively, following reaction with a Δ5 desaturase; and, DHA synthesis requires subsequent expression of an additional C20/22 elongase and a Δ4 desaturase).
In spite of the need for new methods for the production of ARA, EPA and DHA, few Δ9 elongase enzymes have been identified. For example, only a single Δ9 elongase is presently known prior to the Applicants' invention herein. Specifically, PCT Publications No. WO 2002/077213, No. WO 2005/083093, No. WO 2005/012316 and No. WO 2004/057001 describe a Δ9 elongase from Isochrysis galbana and its use (see also GenBank Accession No. AAL37626). Thus, there is need for the identification and isolation of additional genes encoding Δ9 elongases that will be suitable for heterologous expression in a variety of host organisms for use in the production of ω-3/ω-6 fatty acids.
Elongases which have been identified in the past differ in terms of the substrates upon which they act. They are present in both animals and plants. Those found in mammals can act upon saturated, monounsaturated and polyunsaturated fatty acids. However, those found in plants are specific for saturated and monounsaturated fatty acids. Thus, there is a need for a PUFA-specific elongase to produce PUFAs in plants.
The elongation process in plants involves a four-step process initiated by the crucial step of condensation of malonate and a fatty acid with release of a carbon dioxide molecule. The substrates in fatty acid elongation are CoA-thioesters. The condensation step is mediated by a 3-ketoacyl synthase, which is generally rate-limiting to the overall cycle of four reactions and provides some substrate specificity. The product of one elongation cycle regenerates a fatty acid that has been extended by two carbon atoms (Browse et al., Trends in Biochemical Sciences, 27(9):467-473 (September 2002); Napier, Trends in Plant Sciences, 7(2):51-54 (February 2002)).
Based on the utility of expressing Δ9 elongases in conjunction with Δ8 desaturases, there has also been considerable effort to identify and characterize Δ8 desaturases from various sources. Most efforts thus far have focused on the isolation and characterization of Δ8 desaturases from Euglena gracilis; and, several sequence variations of E. gracilis Δ8 desaturases have been reported (see, e.g., Wallis et al., Arch. Biochem. and Biophys., 365(2):307-316 (May 1999); PCT Publication No. WO 2000/34439; U.S. Pat. No. 6,825,017; PCT Publication No. WO 2004/057001; U.S. application Ser. No. 11/166,003 filed Jun. 24, 2005 (PCT Publications No. WO 2006/012325 and No. WO 2006/012326; published Feb. 2, 2006)). More recently, PCT Publication No. WO 2005/103253 (published Apr. 22, 2005) discloses amino acid and nucleic acid sequences for a Δ8 desaturase enzyme from Pavlova salina. Sayanova et al. (FEBS Lett., 580:1946-1952 (2006)) describes the isolation and characterization of a cDNA from the free living soil amoeba Acanthamoeba castellanii that, when expressed in Arabidopsis, encodes a C20 Δ8 desaturase. Also, commonly owned, co-pending application having Provisional Application No. 60/795,810 (filed Apr. 28, 2006) discloses amino acid and nucleic acid sequences for a Δ8 desaturase enzyme from Pavlova lutheri (CCMP459), while commonly owned, co-pending application having U.S. Provisional Application No. 60/853,563 filed Oct. 23, 2006, discloses Δ8 desaturases from the euglenoids Tetruetreptia pomquetensis CCMP1491, Eutreptiella sp. CCMP389 and Eutreptiella cf—gymnastica CCMP1594.
The following commonly owned patent applications relate to the production of PUFAs in oleaginous yeasts (i.e., Yarrowia lipolytica), including: PCT Publication No. WO 2004/101757 and PCT Publication No. WO 2004/101753 (both published Nov. 25, 2004); U.S. application Ser. No. 11/265,761 (filed Nov. 2, 2005; corresponding to PCT Publication No. WO 2006/052870); U.S. application Ser. No. 11/264,784 (filed Nov. 1, 2005; corresponding to PCT Publication No. WO 2006/055322); and U.S. application Ser. No. 11/264,737 (filed Nov. 1, 2005; corresponding to PCT Publication No. WO 2006/052871).
Additionally, PCT Publication No. WO 2004/071467 (published Aug. 26, 2004) concerns the production of PUFAs in plants, while PCT Publication No. WO 2004/071178 (published Aug. 26, 2004) concerns annexin promoters and their use in expression of transgenes in plants; both are commonly owned and copending applications.
Applicants have solved the stated problem by isolating the genes encoding Δ9 elongase from Euglena gracilis and Eutreptiella sp. CCMP389.